1. Field of the Invention
The invention relates to a process for extracting growth factors from growth factor starting material and other cellular components from platelets, platelet rich plasma, and whole blood and, more particularly, to a wound healing and tissue regenerative composition of growth factors and cellular components released from intracellular granules and cellular structures bound by mammalian platelet membranes for use in wound healing and other therapeutic and biomedical uses to repair, regenerate, restore and provide bio-scaffold components to promote and support living cells and tissues and for the purpose of stabilizing or reversing impairment of the normal state of the living animal body or one of its parts whose function has been interrupted or performance of the vital functions compromised by injury, disease, or aging.
2. Description of the Prior Art
The practice of using activated autologous platelets as a treatment in a number of medical and surgical procedures is known, including but not limited to oral and maxillofacial surgery, orthopedic surgery, cosmetic and reconstructive surgery, chronic tissue repair, sports medicine injuries, neurosurgery, cardiovascular surgery, podiatry, hair transplant surgery, immune mediated hair loss, medical research, tissue engineering, and non-surgical cellular therapy. U.S. Pat. Nos. 4,957,742 and 6,649,072 disclose wound healing compositions that include platelet rich plasma (PRP) which prior to use is activated by thrombin to release growth factors from the alpha granules of the platelets.
Extracting therapeutic levels of platelets has been a technical challenge requiring trained cardiovascular perfusionists to operate the equipment originally designed for the production of platelet rich plasma. The clinical practitioner now has access to more simplified equipment that allow processing of PRP with smaller amounts of whole blood in a shorter amount of time. Venous access, clinical expertise, and cost are still challenges that have limited the widespread use of this process throughout the world. Moreover from a commercial standpoint, wound-healing compositions that include platelets must meet costly FDA guidelines applicable to blood products.
Platelet rich plasma (PRP) compositions are being utilized for both human and veterinary applications. PRP formulations may or may not include leucocytes (white blood cells) and are referred to leucocyte-rich PRP or leucocyte-poor PRP. As the knowledge of growth factors expands, a greater understanding of specific growth factors has helped to define their roles with greater precision. In general, while platelets influence anabolic signaling to promote the proliferative and regenerative phases of the healing cascade, leucocytes contain cytokines, a class of growth factors with catabolic activity supportive of an inflammatory response. Thus, a PRP preparation or a growth factor composition can be tailored to the desired anabolic or catabolic activities through selective inclusion or exclusion of leucocytes.
Growth factors are responsible for the wound healing process, as described above. Platelets function as carriers for the growth factors. Growth factors are polypeptides produced by the tissue on which they act. They regulate differentiation, proliferation, migration, and metabolism in target cells, regulating the synthesis of specific adhesion molecules that control cell-cell and cell-substrate interactions. Each GF can have either one or several essential functions for a specific cell, depending on the particular circumstances of the cell environment.
The most widely studied growth factors in relation to tissue proliferation and repair include: bone morphogenetic proteins (BMPs) (eg, BMP-1, BMP-2, and BMP-3); PDGF; insulin-like growth factor (IGFs) (eg, IGF-I and IGF-II); TGFs, especially TGF-8; fibroblast growth factors (FGFs) (ie, acid-FGF and basic-FGF); granulocyte macrophage colony stimulating factor; epidermal growth factor; and VEGF. An important role in repair processes, specifically in the inflammation stages, is also played by cytokines produced by white blood cells, including the interleukins (ILs) IL-1, IL-3, IL-6, and IL-8. All of these growth factors and cytokines act to a greater or lesser extent during the different stages of wound healing which includes tissue necrosis resolution, cell regeneration, cell proliferation and migration, extracellular matrix synthesis, epithelialization, and remodeling.
There is need for an efficient process for extracting and isolating growth factors and platelet granule contents from the platelets contained in plasma for subsequent use in wound healing and for a multitude of bioactive processes supportive of living tissue(s) associated with in vivo or in vitro processes. Preferably, the final product would be a growth factor composition with minimal to no cellular debris.
It is further desirable to prepare a wound healing product, expanded to include processes as described above, that can be subjected to conventional preservation procedures, such as lyophilization, freeze drying, and cryopreservation in a process that does not destroy the growth factors nor the functionality of the growth factor composition. In this manner the shelf life of the product(s) would be significantly prolonged.
In addition to local hemostasis at sites of vascular injury, platelets contain an abundance of growth factors and cytokines that are pivotal in soft tissue healing and bone mineralization. An increased awareness of platelets and their role in the healing process has led to the concept of therapeutic applications.
The preparation of PRP is still dependent on whole platelets, centrifugation or gravity flow or filtration techniques with the exception of cytokine rich plasma (CRP), as discussed in U.S. Pat. No. 8,734,854. During the early years of development, the terminology cytokine rich plasma represented both growth factors and cytokines. More recently growth factors have come to represent positive tissue repair influences and regeneration while cytokines are associated with inflammation, pain, and cleaning up of wounds.
It should be understood that CRP composes both classification types but may be better thought of as complex or complete rich plasma. CRP is unique to PRP in both composition and concentration. Although similar in many ways, the differences are relevant. Intact platelets are common to all forms of PRP. CRP is developed under the influence of sub-atmospheric pressures. Growth factors, cytokines and other intracellular proteins and biochemical components normally released by agonists are extracted from their intracellular positions without the use of agonists. The result: CRP is overwhelmingly acellular.
Production of PRP inherently results in less than 100% of the original content of whole blood cell types of interest. Production of CRP may be derived from 100% of the cellular content of whole blood. Whole blood or various blood cell components, specifically platelets and or white blood cells, are subjected to sub-atmospheric pressures. Maximal levels of each sample's cellular proteins are separated from their intracellular location and released to the extracellular (extracorporeal) fluid environment. Additionally, assays of the sub-atmospheric treated blood led to findings of two peptides not previously recognized and is disclosed in U.S. Pat. No. 8,734,854.
Processing the blood components after being exposed to controlled sub-atmospheric pressure allows for separation of CRP from red blood cells, ghost cells and cell debris resulting in an acellular content. The CRP can then be directly applied to a patient; filtered to remove water thereby concentrating the product into a smaller volume for application in areas where smaller volumes are more efficacious, such as within the confined space of joint, or stored by a unique lyophilization process. Discovered by accident, this specific method of lyophilization preserves biofunctionality, the ability to express bioactive properties, of the CRP components without the addition of fixatives. The latter process remains a trade secret to the inventors.
It has long been the goal to finding a means of preserving or extending the functional longevity of the platelet. Utilizing the concept of negative (sub-atmospheric) pressure, living tissues composed of whole blood and portions thereof were subjected to controlled levels of negative pressures. Under the appropriate range of negative pressure, the blood cell maintains its integrity while undergoing expansion. Too much negative pressure and the cell tears apart. Not enough negative pressure and the cell will not react. With the appropriate degree of negative pressure applied over a defined timeline, the cell expands reducing internal pressures such that particulate matter contained with intracellular alpha, delta (dense) or lambda granules find a pathway of least resistance as they are freed from their confines of the expanding granule membrane. These intra-granule particles are drawn to the lower pressure outside the cell membrane and into the extracellular environment. The contents of the granules are emptied without destroying the integrity of the cell membranes.
The natural life expectancy of a platelet and its granular contents are limited to days using standard blood banking processes. Platelets are cytoplasmic fragments of megakaryocytes lacking nuclei but containing organelles and structures such as mitochondria, microtubules, and three main types of granules. Due to ongoing mitochondrial activity, and mRNA synthesis of proteins, this meets the description of a living cell. These′short-lived cells or cell fragments, whichever one prefers to view them as, have undergone extensive research in order to come up with a reliable means of preservation with retention of normal bioactive potential. For this purpose, platelets will be viewed as cells.
Platelets reside intravascularly. Therefore, any tissue containing a vascular supply will have the same platelet content. The normal concentration of platelets in blood is approximately 140,000 to 400,000 platelets/mm3. These remain in the circulation for about 10 days. Therefore any disruption in one's ability to replace platelets would have rapid and profound effects in the event of a sustained trauma. After tissue injury, platelets are among the first cells to appear and remain in the vicinity of the wound.
The benefits of controlling the collection of and when desirable, the storage of this limited resource, to extend the useful function of a platelet or its contents, would have significant impact on the practice of medicine as it applies to wound healing, regenerative medicine, and other therapeutic and biomedical uses for the purpose of stabilizing or reversing impairment of the normal state of the living animal body or one of its parts whose function has been interrupted or performance of the vital functions compromised by injury, disease, or aging.
Historically, the main purpose of preserving the platelet pertains to correcting bleeding disorders stemming from deficiencies in platelet numbers or platelet function. Advancing technologies have allowed for a growing body of evidence to reveal the critical and diverse role the platelet in the wound healing cascade that includes the steps of hemostasis (clotting), inflammation, proliferation, and regeneration (remodeling) of tissues. Platelets regulate and modulate the rate of tissue repair by releasing biochemical messages, growth factors, that can affect the cell from which it originates (autocrine), or influence local cell activity (paracrine) or distant (endocrine) cell activity. This call to action influences multi-cell activity as well as the migration of healing components to the site of injury.
Over 1200 types of proteins have been identified on or within a platelet. There are approximately 50 to 80 alpha granules per platelet containing greater than 400 different bioactive proteins whose complex interactions in the healing cascade are not yet fully clarified. Dense bodies or Delta granules (250-300 nm) contain ATP, proaggregatory factors such as adenosine 5′-diphosphate (ADP), calcium, and 5-hydroxytryptamine (serotonin), pyrophosphate, histamine and other factors which promote adhesion of platelets and cause vasoconstriction. Lambda granules (175-250 nm) contain lysosomal, proteases, lipases, nucleases and polysaccharidases. These bioactive enzymes function to remove infectious agents and cellular debris.
It should be understood that PRP is more than just platelets and that it contains many bioactive factors that act in anabolic, catabolic, proinflammatory, and anti-inflammatory pathways. Some components are also involved in the modulation of the immune response. The precise combination and concentration of platelets, leukocytes, and other plasma components best for musculoskeletal healing are not presently known, and clinicians should be aware that the effects of PRP are not solely based on platelet concentration. A maximal efficacious concentration beyond which the platelet concentration will provide no further clinical benefits likely exists. Although the effects of many of the proteins in PRP on musculoskeletal tissues are still unknown, they likely contribute to the biologic healing process. Finally, it is imperative for individuals involved in clinical study design and all clinicians to take into consideration diurnal variation in platelet count and that, simply, generation of PRP will fail in some patients in some instances.
Activation of a platelet by an agonist, such as thrombin, collagen, or other agonist known in the art, leads to the differential release of granule material from within the platelet. Such granulation activation (differential degranulation) results in the specific and sequential release of groups of growth factors and other granular contents over time.
Physical agonists such as rapid cooling and freezing temperatures as used in standard lyophilization, cryopreservation, and freeze drying processes result in a percentage of platelet destruction and a significant loss of bioactivity rendering them less potent. The appropriate application of sub-atmospheric pressure results in a rapid and thorough release of granular contents with retention of bioactivity.
The platelet cytoplasm contains two distinct pathways: first a closed dense tubular system which does not open to the cell membrane and second an open cannicular system which appears to be an invagination of the outer cell membrane and does open to the cell membrane and through which platelet granular contents are dispersed when activated. Where the dense tubular system lies adjacent to the open cannicular system, transfer of proteins across membranes will occur. During the unconventional application of a sub-atmospheric pressure without the use of natural or known platelet activators, findings support that negative pressures assist in the nonspecific extraction (release) and discharge of granular contents into the dense tubular system and or cannicular system of the platelet from which growth factors are released beyond the cell membrane to the extracellular (extracorporeal) environment. In effect, a rapid and complete, non-differential or nonspecific release of granular material results from a sufficient, application of sub-atmospheric pressure.
Membranous pseudopod formation is discussed at length in the medical literature. Natural or agonist induced activated platelets undergo pseudopod formation. Studies are lacking as to whether or not sub-atmospheric pressure will induce pseudopod formation but it is hypothesized that it will not occur with rapidly applied negative pressure. Rather the entire platelet cell will expand more uniformly.
Human platelet granules degranulate via the intracellular caninicular system and dense tubular system. Equine and bovine platelet granules have been shown to release growth factors directly from the cellular membrane. Canine platelet degranulation appears to vary between cell membrane and cannicular system. Various forms of disease have been studied that have an affect on the platelet degranulation process.
When applied to existing wounds, growth factors are known to attract more platelets (proaggregatory factors), macrophages, regenerative cells such as mesenchymal stem cells and osteoblasts, and increase the rate of collagen laydown, vascular ingrowth, fibroblast proliferation and overall healing. The release of a protein known as platelet-derived growth factor (PDGF) serves as a chemotactic signal for monocytes, neutrophils and fibroblasts, which then move into the wound to begin the inflammatory stage of the healing process. During this time, monocytes secrete a number of factors, including PDGF and transforming growth factor-beta 1 (TGF-β1) also found in platelets. In this manner fibroblasts are activated to begin the repair or regenerative stage of the healing process. Subsequently, wound healing continues through the process of collagen remodeling within the wound.
Platelet rich plasma compositions are being utilized for both human and veterinary applications. PRP formulations may or may not include leucocytes (white blood cells) and are referred to leucocyte-rich PRP or leucocyte-poor PRP. As the knowledge of growth factors expands, a greater understanding of specific growth factors has helped to define their roles with greater precision. In general, while platelets influence anabolic signaling to promote the proliferative and regenerative phases of the healing cascade, leucocytes contain cytokines, a class of growth factors with catabolic activity supportive of an inflammatory response to help resist infection and remove cellular and tissue debris. Thus, a PRP preparation or a growth factor composition can be tailored to the desired anabolic or catabolic activities through selective inclusion or exclusion of leucocytes.
Growth factors are responsible for the wound healing process, as described above. Platelets function as carriers for the growth factors. This further understanding is clearly recognized and included with use of the terminology “growth factor composition” which is understood to include the contents of platelet alpha, dense, and lambda granules that contain over 400 different bioactive proteins and biochemicals whose complex interactions in the healing process are not yet fully clarified as well as components of the extracellular fluid or plasma. Therefore, there is a desire for an efficient process for extracting and isolating growth factors and when appropriate, additional bio-substances, from the platelets and the blood, plasma, or tissue(s) containing the platelets at the time of application of the sub-atmospheric pressure for subsequent use in wound healing and for a multitude of bioactive processes supportive of living tissue associated with in vivo or in vitro processes. The use of a natural growth factor composition as part of a supportive medium or bioscaffold to produce whole organs or tissues in vitro has significant implications. Preferably, the final product shall be free of other selective components that are typically found in conventional platelet enriched wound healing products, namely whole platelets, ghost platelets, white blood cells, red blood cells, bacteria, and other cellular debris.
There is further benefit to preparing naturally derived wound healing products that can be subjected to conventional preservations, such as lyophilization, freeze drying, and cryopreservation in a process that does not destroy the growth factors nor the functionality of the growth factor composition. In this manner the shelf life of the product(s) would be significantly prolonged.
Therefore, there is a need for a process for isolating and extracting growth factors in a non-destructive manner from platelets. The resulting composition may selectively contain other platelet and if desired platelet rich plasma components or, may or may not be substantially free of other components, such as whole platelets, ghost platelets, white blood cells, red blood cells and bacteria, and can be used fresh for immediate use or lyophilized for delayed use into a shelf-stable, non-refrigerated product for subsequent use.